Method for Preparing Peptides

ABSTRACT

The invention relates to a method for preparing peptides comprising the step of forming a peptide bond wherein the carboxyl group of a first amino acid or first peptide is activated and an amino group of the first activated amino acid or first peptide is protected by a protecting group having a water-solubility enhancing group and the activated carboxyl group of the first amino acid or first peptide is reacted with an amino group of a second amino acid or second peptide wherein said carboxyl group of the first amino acid or first peptide is activated in the absence of the second amino acid or second peptide. The invention further relates to peptides comprising a protecting group having a water-solubility enhancing group being bound to the amino group and an activated or free carboxyl group.

The present invention relates to an improved method for preparingpeptides, and to peptides being obtainable by the method.

Peptides are linked chains of amino acids and represent the precursorsof proteins. Peptides and proteins are the elementary components of allliving systems and are involved in a variety of processes of life. Theyhave many applications in medical and biological sciences. As aconsequence, the capability to synthesize peptides and proteins is ofhigh significance to human life.

Hence, the synthetic production of peptides is of significant interest.Peptides are conventionally synthesized by coupling the carboxyl group(C-terminus) of one amino acid to the amino group or N-terminus ofanother.

In general, the chemical synthesis starts from the C-terminal amino acidof the chain. In addition, it requires an additional chemical group toprotect the C-terminus of the first amino acid. The process of couplingindividual amino acids can be accomplished by employment of differentchemical coupling reagents, resulting in an activated carboxyl group.After formation of the amide bond, the Nα-protecting group is removed;conditions for this are depending on the used protecting group. However,solution phase chemistry is slow and labour-intensive because theproduct has to be isolated from the reaction solution manually aftereach step by a purification step (extraction, washing orcrystallisation). Despite the disadvantages of the method, solutionphase chemistry is still the method of choice for production of shortpeptides, for example di-, tri- or tetrapeptides, longer sequences arepossible with fragment condensation procedures. That's because Boc orCBz protected amino acids are less expensive raw materials thanFmoc-amino acids, and the solution phase chemistry has nearly unlimitedsynthesis capacity.

Solid-phase peptide synthesis (SPPS) was introduced by Merrifield et al.in 1963 with the intent to overcome the intermediate purificationproblems associated with peptide assembly in solution (see Stewart andYoung, Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed.,1984), Chan and White, Fmoc Solid Phase Peptide Synthesis: A PracticalApproach (Oxford University Press, 2000)). Upon solid-phase synthesis,amino acids are consecutively coupled to result in a peptide possessingthe desired sequence while the C-terminus is anchored to an insolublepolymeric support (solid phase). Once the desired sequence has beenassembled, the peptide is cleaved from the solid support.

This synthetic scheme requires the protection of the α-amino group ofthe incoming amino acid in order to avoid self-polymerization. Thestandard protecting groups for α-amino functions are the acid-labiletert-butyloxycarbonyl (Boc) group, the base-labilefluorenylmethyloxycarbonyl (Fmoc) group and the allyloxycarbonyl (Alloc)group which is removed under neutral conditions with Pd catalysis in thepresence of PhSiH₃ as scavenger for the allyl system.

Methods for solid-phase peptide synthesis following any of the threeabove-mentioned α-amino protection schemes generally require additionalprotection of reactive side chains of the constituent amino acids fromunwanted chemical transformations. Therefore, it is necessary that theseprotecting groups are resistant to the agents used during the couplingcycle. Additionally, the linkage between the growing peptide chain andthe solid-phase support has to be stable towards the conditions ofα-amino deprotection and chain assembly.

In the case of the Fmoc-based α-amino protection, the side-chain groupsshould be resistant to the basic reagents used to remove the Fmocmoiety. The side-chain protecting groups are generally removed by mildacidic reagents after the peptide chain has been assembled. Theseside-chain protecting groups are generally cleaved by anhydrous HF,trifluoromethanesulfonic acid or trifluoro acetic acid (TFA) after thedesired peptide chain has been assembled.

The peptide assembly procedure is typically performed in polar aproticorganic solvents such as dimethyl formamide (DMF), N-methylpyrrolidone(NMP), dimethyl sulfoxide (DMSO) and dichloromethane (DCM), or a mixtureof these organic solvents because of the strong hydrophobic character ofthe α-amino protecting groups Fmoc and Boc, which are insoluble in waterand which are frequently used in SPPS. Additionally, the side-chainprotecting groups commonly used in SPPS are usually hydrophobic andrender the amino acid insoluble in water.

SPPS approaches applying Fmoc and Boc protection are widely used butsuffer from the need for the previously mentioned organic solvents whichare costly and toxic. DMF, for example, comes with considerable healthand environmental risks; it has been linked to cancer in humans, and itis suspected to cause birth defects. Hence, the use of these toxicsolvents requires special technical equipment and precautions, as e.g.performing the reaction under the fume hood and handling by highlyspecialized personnel. In addition, the disposal of the used solvent isproblematic and expensive. As a consequence, SPPS using organic solventsis expensive and restricted to specialised laboratories with specialequipment for organic chemistry synthesis.

As an attempt to overcome this problem, Hojo et al. proposed the use ofwater-soluble protecting groups (Chem. Pharm. Bull. 2004, 52, 422-427and Tetrahedron Lett. 2004, 45, 9293.) They developed several protectinggroups for this purpose, among them2-(Phenyl(methyl)sulfonyl)ethyloxycarbonyl tetrathioborate (Pms),Ethanesulfonylethoxycarbonyl (Esc), and 2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps). In WO 2013 115813 A1 the CEM Corporation claims thedeprotection of α,β-unsaturated sulfones in water or aqueous systems andtheir usage in water-based SPPS.

Furthermore, WO 2016/050764 A1 describes a water-based peptidesynthesis.

The methods mentioned above can be used to synthesize peptides. However,it is a permanent desire to improve these methods.

Therefore, it is an object of the invention to provide an improvedmethod for peptide synthesis in general. The method should be applicableto liquid and solid-phase peptide synthesis.

According to a specific object of the present invention, the methodshould provide a high yield. Furthermore, the synthesis of the peptideshould be performed at low costs. A further object of the presentinvention is a method having a low racemization of the amino acid unitsbeing incorporated into the peptide chain. In addition, the method forpreparing peptides should be executed with low waste and with highenvironmental sustainability.

Furthermore, the present method should be performable using knownapparatus in order to achieve a high acceptability. Moreover, the methodshould have a low impact on the environment. Preferably, onlyenvironmentally acceptable solvents and compounds should be used and/orthe solvents should be recovered easily that no critical impact to theenvironment should be expectable.

These objects and further objects which are not stated explicitly butwhich are immediately derivable or discernible from the connectionsdiscussed herein by way of introduction are solved by a method forpreparing peptides comprising the step of forming a peptide bond havingall features of claim 1.

The present invention accordingly provides a method for preparingpeptides comprising the step of forming a peptide bond characterized inthat the carboxyl group of a first amino acid or first peptide isactivated and an amino group of the first activated amino acid or firstpeptide is protected, preferably by a protecting group having awater-solubility enhancing group or by a solid phase, and the activatedcarboxyl group of the first amino acid or first peptide is reacted withan amino group of a second amino acid or second peptide wherein saidcarboxyl group of the first amino acid or first peptide is activated inthe absence of the second amino acid or second peptide.

It is thus possible in an unforeseeable manner to improve the prior artmethods for preparing peptides as mentioned above.

The peptides can be obtained very inexpensively. Surprisingly, thepeptides obtained contain only very small amounts of by-products,especially a low racemization of the amino acid units being incorporatedinto the peptide chain can be achieved by the present method.

In addition, the process according to the invention enables aparticularly selective preparation of the peptides. Furthermore, themethod according to the invention can be performed in a simple andinexpensive manner, the product being obtainable in high yields and,viewed overall, with low energy consumption.

In addition, the method for preparing peptides can be executed with lowwaste and with high environmental sustainability.

Furthermore, the present method can be performable using known apparatusin order to achieve a high acceptability. Moreover, the methods have alow impact on the environment. Preferably, only environmentallyacceptable solvents and compounds are used and/or the solvents can berecovered easily such that no critical impact to the environment isexpectable.

According to a first embodiment of the invention, an amino group of thefirst activated amino acid or first peptide is protected by a protectinggroup having a water-solubility enhancing group. In addition thereto,the first amino acid or first peptide preferably comprises additionalprotecting groups. Preferably, the functional group to be protected ispreferably selected from amine, alcohol, thiol, carboxyl, phosphonoand/or seleno groups.

In preferred embodiments the water-solubility enhancing functional groupis selected from the charged functional groups SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester and N(CH₃)₃ ⁺. Using charged functional groups asthe water-solubility enhancing functional group has been proven usefulfor increasing solubility in environmentally friendly solvents, e. g.water-solubility more efficiently than the uncharged functional groups.

According to a second embodiment of the invention, an amino group of thefirst activated amino acid or first peptide is protected by solid phase.Protection by a solid phase is preferably achieved by a covalent bond ofthe amino group of the first activated amino acid or first peptide to asolid phase. Preferably, the covalent bond of the amino group of thefirst activated amino acid or first peptide to the solid phase iscleavable. Consequently, the peptide or protein achieved with thepresent method can be released from the solid phase by a cleavagereaction as described in more detail below. In addition thereto, thefirst amino acid or first peptide preferably comprises additionalprotecting groups. Preferably, the functional group to be protected ispreferably selected from amine, alcohol, thiol, carboxyl, phosphonoand/or seleno groups.

The protection of the amino group of the first activated amino acid orfirst peptide by a protecting group having a water-solubility enhancinggroup is preferred over a protection using a solid phase in view ofsustainability, costs and efficiency. On the other hand, a protection ofthe amino group of the first activated amino acid or first peptide by asolid phase provides improvements regarding the production of peptideshaving a high number of amino acid units.

Preferably, the forming of the peptide bond is achieved using anenvironmentally friendly solvent. The forming of the peptide bondincludes the activation of the carboxyl group of said first amino acidor first peptide, the reaction of the activated carboxyl group of thefirst amino acid or first peptide with an amino group of a second aminoacid or second peptide or both steps. Conventionally, peptide synthesisis achieved in solvents which are critical to the environment or health.These solvents include dimethylformamide (DMF) andN-methyl-2-pyrrolidone (NMP). Although these solvents can be used toperform the present method, the present invention is not limited theretobut environmentally friendly solvents can be used which provides furthercost advantages and lower precautions based on environmental standards.Therefore, one of the advantages of the invention is the utilization ofan amino acid and/or peptide that is soluble in an environmentallyfriendly solvent in its protected form.

As used herein, the term “soluble in an environmentally friendly solventin its protected form” means that the composition has the degree ofsolubility necessary for the desired reaction to proceed in a solventsystem. As in the case with any composition, the term “soluble” does notimply unlimited solubility in any or all amounts.

Preferably the environmental friendliness of the solvents is based ontheir impact on health or the environment. Substances that are listed bythe European Chemicals Agency (ECHA) as substances of very high concern(SVHCs) or on the candidate list of substances of very high concern arenot classified as environmentally friendly solvents. In order to avoidusing harmful organic solvents that may have serious and oftenirreversible effects on human health and the environment, all solventslisted by the ECHA as SVHCs should be avoided (state 14 Nov. 2017).

According to a preferred embodiment, the environmentally friendlysolvent preferably has a DNEL value of at least 5 mg/m³, more preferablyat least 10 mg/m³, more preferably of at least 20 mg/m³, more preferablyat least 40 mg/m³, more preferably at least 45 mg/m³, more preferably atleast 60 mg/m³, more preferably at least 100 mg/m³, more preferably atleast 120 mg/m³, and even more preferably of at least 200 mg/m³(inhalation, systemic, as provided by the ECHA state 14 Nov. 2017).

In a specific embodiment, said environmentally friendly solventpreferably has a lethal dose LD50 of at least 0.1 g/kg, preferably of atleast 0.5 g/kg, more preferably of at least 1.0 g/kg, more preferably ofat least 1.5 g/kg, more preferably of at least 2 g/kg and even morepreferably of at least 2.5 g/kg (oral, rat).

Preferred solvents, more preferably environmentally friendly solventsinclude protic solvents, such as water, primary, secondary and/ortertiary alcohols, non-protic solvents such as ketones, nitriles,lactones, lactams, carbon acid amides, carbon acid ester, ether, ureaderivatives, sulfoxides, sulfones, carbonate ester. Preferred examplesof non-protic solvents are e. g. dimethylacetamide, ethyl methyl ketone,acetone, methyl isopropyl ketone, methyl isobutyl ketone, methyl n-butylketone, methyl t-butyl ketone, methyl isoamyl ketone, dimethylsulfoxide, hexamethylphosphoric triamide, tetrahydrofuran, dioxane,dimethoxyethane, diethylene glycol, dimethyl ether, ethyl acetate,tertiary butyl acetate, or mixture(s) thereof.

A protic solvent is a solvent that has a hydrogen atom bound to anoxygen (as in a hydroxyl group) or a nitrogen (as in an amine group).Non-protic solvents are solvents which do not include a hydrogen atombound to an oxygen (as in a hydroxyl group) or a nitrogen (as in anamine group). Therefore, a ketone is a non-protic solvent although someisomers (enol isomer) may include such hydrogen atoms. However, based onthe low percentage of these isomers being formed, these isomers have tobe neglected with regard to the present invention.

The present invention can be achieved using a protic solvent, such aswater, primary, secondary and/or tertiary alcohols. Primary andsecondary amines should not be used as a solvent based on the couplingreaction for forming a peptide bond. Preferred alcohols includemethanol, ethanol, isopropanol, 2-propanol, n-propanol, n-butanol,isobutanol, sec-butanol, tert-butanol, or a mixture thereof. Proticsolvents provide advantages regarding costs and lower precautions basedon environmental standards. Regarding protic solvents water isespecially preferred. In addition to the costs advantages and highenvironmental acceptance, water provides improvements regarding safety,especially low flammability and combustibility can be achieved bysolvents comprising water. Furthermore, water provides higher yield inview of other protic solvents such as alcohols, especially primaryalcohols.

In an embodiment of the present invention, the solvent, preferably theenvironmentally friendly solvent preferably comprises at least 5% byweight, more preferably at least 10% by weight, even more preferably atleast 25% by weight of a protic solvent, preferably water based on thetotal weight of the solvent, preferably the environmentally friendlysolvent.

In a further embodiment of the present invention, the solvent,preferably the environmentally friendly solvent preferably comprises atleast 5% by weight, more preferably at least 10% by weight, even morepreferably at least 25% by weight of a protic solvent, preferably water,based on the total weight of the solvent, preferably the environmentallyfriendly solvent.

Surprising improvements can be achieved by a solvent, preferably anenvironmentally friendly solvent comprising a non-protic organic solventand/or a secondary and/or tertiary alcohol, preferably non-proticorganic solvent and/or a tertiary alcohol, more preferably a non-proticorganic solvent. The use of a non-protic organic solvent and/or asecondary and/or tertiary alcohol provides astonishing improvements inyield. Furthermore, the use of non-protic solvents provides a higherdegree of conversion. Therefore, longer peptide chains are achievable ata considerable yield. Furthermore, the activation of an amino acid orpeptide as mentioned above and below is easier and the activated aminoacid or peptide is more stable. Therefore, lower amounts of activationagent can be used. In addition, further advantages can be achieved ifthe method is performed on an ion exchanger as mentioned above andbelow. Preferably, the ion exchanger is used in a method wherein anamino group of the first activated amino acid or first peptide isprotected by a protecting group having a water-solubility enhancinggroup. Using a non-protic organic solvent and/or a secondary and/ortertiary alcohol in combination with an ion exchanger providesimprovements in yield of the peptides prepared. These improvements canbe increased by increasing the amount of non-protic organic solvent.These improvements are achieved by using a solvent, preferably anenvironmentally friendly solvent comprising a non-protic organic solventand/or a secondary and/or tertiary alcohol, preferably non-proticorganic solvent and/or a tertiary alcohol, more preferably a non-proticorganic solvent for the activation of the carboxyl group of said firstamino acid or first peptide. Furthermore, these improvements areachieved by using a solvent, preferably an environmentally friendlysolvent comprising a non-protic organic solvent and/or a secondaryand/or tertiary alcohol, preferably non-protic organic solvent and/or atertiary alcohol, more preferably a non-protic organic solvent for thereaction of the activated carboxyl group of the first amino acid orfirst peptide with an amino group of a second amino acid or secondpeptide as mentioned above and below. In a very preferred embodiment,the activation of the carboxyl group of said first amino acid or firstpeptide and the reaction of the activated carboxyl group of the firstamino acid or first peptide with an amino group of a second amino acidor second peptide bond as mentioned above and below are achieved byusing a solvent, preferably an environmentally friendly solventcomprising a non-protic organic solvent and/or a secondary and/ortertiary alcohol, preferably non-protic organic solvent and/or atertiary alcohol, more preferably a non-protic organic solvent.

In an embodiment of the present invention, the solvent, preferably theenvironmentally friendly solvent preferably comprises at least 5% byweight, more preferably at least 10% by weight, even more preferably atleast 25% by weight of a non-protic organic solvent and/or a secondaryand/or tertiary alcohol, preferably non-protic organic solvent based onthe total weight of the solvent, preferably the environmentally friendlysolvent.

In a particularly appropriate variant said solvent, preferably saidenvironmentally friendly solvent comprises a non-protic organic solventas a first solvent and water and/or alcohol as a second solvent,preferably a non-protic organic solvent and water.

In preferred embodiments said solvent, preferably said environmentallyfriendly solvent comprises 5 to 95% by weight, preferably 10 to 70% byweight of said second solvent, based on the total weight of the solvent,preferably the environmentally friendly solvent. Furthermore, saidsolvent, preferably said environmentally friendly solvent preferablycomprises 5 to 95% by weight, more preferably 10 to 90% by weight, evenmore preferably 30 to 70% by weight of said first solvent, based on thetotal weight of the solvent, preferably the environmentally friendlysolvent.

Surprisingly, the use of non-protic organic solvents having a lowpolarity such as dimethylacetamide, ethyl methyl ketone, acetone, methylisopropyl ketone, methyl isobutyl ketone, methyl n-butyl ketone, methylt-butyl ketone, methyl isoamyl ketone, dimethyl sulfoxide,hexamethylphosphoric triamide, tetrahydrofuran, dioxane,dimethoxyethane, diethylene glycol, dimethyl ether, ethyl acetate,tertiary butyl acetate, or mixture(s) thereof and/or protic solventssuch as water and alcohols provide astonishing improvements regardingthe racemization of the peptides formed. In contrast thereto the use ofpolar aprotic organic solvents, such as dimethylformamide (DMF) andN-methyl-2-pyrrolidone (NMP) provide a higher racemization of thepeptides formed. Preferably, the polarity is defined as the dipolemoment of the molecule. Preferably, the dipole of the aprotic organicsolvent is at most 12.6 10⁻³⁰ Cm, more preferably at most 12.5 10⁻³⁰ Cm,preferably measured at 25° C. and 1023 mbar by the measurement of thedielectric constant using the Debye equation and/or measuring the Starreffect, preferably by measuring the dielectric constant. In a preferredembodiment, the racemization of the peptides formed can be avoided orreduced by adding and/or using a protic solvent, more preferably water.The addition of a protic solvent, preferably water reduces theracemization of the peptides formed if a polar aprotic organic solventis used.

Preferably, the forming of the peptide bond is achieved in solutionhaving no strong basic condition, preferably at a pH below 12, morepreferably at a pH below 10 measured by adding water to the solution at25° C. according to the professional methods known, e.g. pH-electrodes.The measured sample preferably comprises at least 80% by weight water.Preferably, pH-electrodes are calibrated using two, three or more buffersolutions having a specified pH value. Performing the reaction insolution having no strong basic condition provides astonishingimprovements regarding the racemization of the peptides formed.

According to the invention, the carboxyl group of a first amino acid orfirst peptide is activated and the activated carboxyl group of the firstamino acid or first peptide is reacted with an amino group of a secondamino acid or second peptide.

In a preferred embodiment carboxyl group of a first amino acid or firstpeptide is activated by a coupling agent.

Preferably, the coupling agent is a combination of a carbodiimide,preferably diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimid (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC),2-(((ethylimino)methylene)amino)-2-methylpropane-1-sulfonate (ESC) and2,2′-(methanediylidenebis(azanylylidene))bis(2-methylpropane-1-sulfonate) (DSC), and an active ester formingcompound, preferably 1-hydroxybenzotriazole (HOBt),1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (NHS),ethylcyano(hydroxyimino)acetate (Oxyma),hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt),4-dimethylaminopyridine (DMAP), 2-Hydroxypyridine-N-oxide (HOPO).Coupling agent could also be based on phosphonium- and theaminium-(imonium-) type reagents such as(benzotriazol-1-yloxy)tris(dimethylamino) phosphoniumhexafluorophosphate (BOP), (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP),bromotri(pyrrolidino)phosphonium hexafluorophosphate (PyBrOP), ethylcyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphoniumhexafluorophosphate (PyOxim),(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (DEPBT),(1H-benzotriazol-1-yloxy)-N,N-dimethylmethaniminium hexachloroantimonate(BOMI), 5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrroliumhexachloroantimonate (BDMP),5-(7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrroliumhexachloroantimonate (AOMP),5-(pentafluorophenyloxy)-3,4-dihydro-1-methyl-2H-pyrroliumhexachloroantimonate (FOMP),5-(3′,4′-dihydro-4′-oxo-1′,2′,3′-benzotriazin-3′-yloxy)-3,4-dihydro-1-methyl2H-pyrrolium hexachloroantimonate (DOMP),1-(1H-benzotriazol-1-yloxy)phenylmethylene pyrrolidiniumhexachloroantimonate (BPMP),5-(succinimidyloxy)-3,4-dihydro-1-methyl-2H-pyrroliumhexachloroantimonate (SOMP), tetramethylfluoroformamidiniumhexafluorophosphate (TFFH),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU), O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate(HATU), (2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium)hexafluorophosphate (HCTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate(TATU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TNTU),1-((dimethyl-amino)(dimethyliminio)methoxy)-2-hydroxypyridiniumtetrafluoroborate (TPTU),O-(cyano(ethoxycarbonyl)methylenamino)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TOTU),2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TDBTU),1-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]uroniumhexafluorophosphate (COMU),4-{[1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)ylidenaminooxy](dimethylamino)-methylen}morpholin-4-iumhexafluorophosphat(COMBU),N-{[1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(6H)-ylidenaminooxy](dimethylamino)methylen}-N-methylmethanaminiumhexafluorophosphat(TOMBU), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate(TSTU). Also miscellaneous coupling reagents like EEDQ(N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) and DMTMM(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salts) andother condensation products of substituted cyanuric chloride with bases.

In a preferred embodiment, a carboxyl group of a first amino acid orfirst peptide is activated by a coupling agent. Preferably, an excess ofcoupling agent is used, preferably the coupling agent is used in atleast two-fold molar amount, more preferably in at least three-foldmolar amount in relation to the first amino acid or first peptide. Anexcess of coupling agent is preferably removed from the reaction mixturebefore the second amino acid or second peptide is added to the activatedfirst amino acid or first peptide. In an alternative embodiment, thecoupling agent is used in an equimolar amount or below in order toachieve that all of the added coupling agent react with the first aminoacid or first peptide. In that case, no excess of coupling agent remainsin the reaction mixture.

In an embodiment of the present invention, it can be provided that thefirst amino acid or first peptide is preferably ionically contacted withan ion exchanger. The contacting of the first amino acid or firstpeptide can be achieved at any step of the present method as mentionedabove and below. Preferably, the first amino acid or first peptide beingionically contacted with an ion exchanger comprises at least oneprotecting group having a water-solubility enhancing group, morepreferably an ionic water-solubility enhancing group.

In a preferred embodiment, the carboxyl group of a first amino acid orfirst peptide is preferably activated by a coupling agent while thefirst amino acid or first peptide is ionically bound to an ionexchanger. Preferably, the first amino acid or first peptide beingionically bound to an ion exchanger comprises at least one protectinggroup having a water-solubility enhancing group, more preferably anionic water-solubility enhancing group. In a variant of the presentmethod, excess of coupling agent is preferably removed from the ionexchanger before a second amino acid or second peptide is added to theion exchanger.

In a further preferred embodiment, said forming of a peptide bond isachieved while an amino acid or a peptide is ionically bound to an ionexchanger. Preferably, the first amino acid or first peptide beingionically bound to an ion exchanger comprises at least one protectinggroup having a water-solubility enhancing group, more preferably anionic water-solubility enhancing group. The ionically bounding to theion exchanger may include any step of forming the peptide bondcomprising the activation of the carboxyl group of an amino acid orpeptide, the reaction of the activated carboxyl group of an amino acidor peptide with an amino group of another amino acid or another peptideor both steps. Preferably, said forming of a peptide bond is achievedwhile an amino acid or peptide is not covalently bound to an ionexchanger.

According to the second embodiment of the present invention, the aminogroup of the first activated amino acid or first peptide is protected bysolid phase. The solid phase (solid support) is preferably an insolubleresin, preferably being based on crosslinked polystyrene, polyacryl,polyphenol, polysaccharide, polyamide or polylysine. Preferably, thecovalent bonding of the amino group of the first activated amino acid orfirst peptide to the solid support is achieved by a linking group. Thelinking group preferably provides a cleavable link of the growingpeptide to the solid phase. The cleavage is preferably achieved using acleaving composition as mentioned below. The use of solid supports forpreparing peptides is well known in a synthesis wherein the C-terminusof the first amino acid is anchored via the N-term inus and furtheramino acids are coupled to the N-terminus of the peptide being anchoredto the solid support. Solid supports having reactive groups which canfrom a covalent bond with the amino group of the first activated aminoacid or first peptide are commercially available. The reactive groups ofthe solid phase being useful for forming a linkage with the amino groupof the first activated amino acid or first peptide are well known in theart. The person skilled in the art will choose the groups depending onthe reaction conditions and the required stability and cleavability ofthe bonding.

Preferably, a carboxyl group of the second amino acid or second peptidebeing reacted with said first activated amino acid or first peptide isnot protected. In a preferred embodiment, the protection of a carboxylgroup of the second amino acid or second peptide can be avoided. Suchembodiment provides astonishing cost advantages. Protection may beachieved by a protection group or by bonding to a solid phase.

In a preferred embodiment, the synthesis of a peptide is build up viathe carboxyl group of an amino acid or peptide and a carboxyl group ofthe second amino acid or second peptide being reacted with said firstactivated amino acid or first peptide is not protected wherein themethod comprises the steps of activating a first amino acid or firstpeptide, removing an excess of coupling agent, adding a second aminoacid or second peptide to the first activated amino acid or firstpeptide and reacting the second amino acid or second peptide with thefirst activated amino acid or first peptide and forming a peptide bond,removing residues of the reaction by a washing step. These steps can berepeated in order to achieve a desired peptide.

In an alternative embodiment, the synthesis of a peptide is build up viathe carboxyl group of an amino acid or peptide and a carboxyl group ofthe second amino acid or second peptide being reacted with said firstactivated amino acid or first peptide is protected wherein the methodcomprises the steps of activating a first amino acid or first peptide,adding a second amino acid or second peptide having a protected carboxylgroup to the first activated amino acid or first peptide and reactingthe second amino acid or second peptide with the first activated aminoacid or first peptide and forming a peptide bond, optionally cappingunreacted carboxyl groups of first amino acid or first peptide,deprotecting the carboxyl group of the second amino acid or secondpeptide. Optionally residues of the reaction are removed by a washingstep being performed between the steps mentioned. These steps can berepeated in order to achieve a desired peptide.

It is preferred that said second amino acid or second peptide ispreferably a peptide having at least two amino acid units, morepreferably a peptide having at least four amino acid units, even morepreferably a peptide having at least six amino acid units. Suchembodiment provides unforeseeably improvements regarding yield of thepeptides being prepared and provides cost improvements.

According to a preferred embodiment, the amino group of the firstactivated amino acid or first peptide is protected by a protecting grouphaving a water-solubility enhancing group being ionically bound to anion exchanger, preferably the water-solubility enhancing group comprisesan ionic group being ionically bound to an ion exchanger.

The type of the protecting group being used in order to achieve an ionicbound to the ion exchanger is not critical.

Preferably, the ion exchanger is an ion exchange resin, preferably beingbased on crosslinked polystyrene, polyacryl, polyphenol orpolysaccharide and having a functional group or the ion exchanger is amineral ion exchanger, preferably based on silica.

Preferably, said functional group of the ion exchange resin is a tetraalkyl ammonium group (—NR₃ ⁺), a primary, secondary or tertiary ammoniumgroup (—NH₂, —NHR, —NR₂), a carboxylic group (—COO⁻), sulfonic group(—SO₃ ⁻), wherein R is an alkyl group having 1 to 10 carbon atoms.

In the present description, conventional terms regarding the descriptionof the ion exchanger are used. Consequently, a basic ion exchanger is asolid having the ability to exchange anions. An acidic ion exchanger isa solid having the ability to exchange cations.

In a specific embodiment, a basic exchange resin is used and the aminoacid or peptide comprises an anionic water-solubility enhancing group,preferably weak basic exchange resin is used comprising primary,secondary and/or tertiary amino groups. Preferably, an anionic ionexchanger is used.

In another embodiment of the present invention, a cationic ion exchangeris used. Preferably an acid exchange resin is used and the amino acid orpeptide comprises a cationic water-solubility enhancing group.

The present amino acid or peptide preferably comprises a protectinggroup having a water-solubility enhancing group. The expression“water-solubility enhancing group” describes a group providing improvedwater solubility to a protecting group. In principle every conventionalprotecting group being used in peptide synthesis can be used which havea group or a modification providing improved water-solubility. Theseprotecting groups having a water-solubility enhancing group includecompounds as mentioned by Hojo et al. proposed (Chem. Pharm. Bull. 2004,52, 422-427 and Tetrahedron Lett. 2004, 45, 9293.) and in WO 2013 115813A1, as e.g. 2-(phenyl(methyl)sulfonyl)ethyloxycarbonyl tetrathioborate(Pms), ethanesulfonylethoxycarbonyl (Esc), and2-(4-sulfophenylsulfonyl)ethoxy carbonyl (Sps).

In a preferred embodiment, the protecting group having awater-solubility enhancing group comprises a back bone structure and alinking group being derived from a reactive group.

In a specific embodiment, the protection of said amino acid or peptideis preferably achieved by reacting an amino acid or a peptide with aprotective agent comprising

I. a backbone structure,II. at least one water-solubility enhancing group andIII. at least one reactive group,wherein the backbone structure comprises a moiety selected from thegroup consisting of 9-methylfluorene, t-butane and/or mono-, di ortriphenylmethane,wherein the water-solubility enhancing group is selected from the groupconsisting of SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, CN, OSO₃ ⁻ ester, OPO₃²⁻ ester and combinations thereof, and wherein the water-solubilityenhancing group and the reactive group are attached to the backbonestructure via at least one covalent bond.

The protective agent may comprise one or more water-solubility enhancingfunctional groups, in preferred embodiments the protective agentcomprises more than one water-solubility enhancing functional group. Infurther preferred embodiments the protective agent comprises at leasttwo water-solubility enhancing functional groups. In other preferredembodiments the protective agent comprises from 1 to 8, 2 to 7, or 3 to4 water-solubility enhancing functional groups. The inventors have foundout that it may be useful to have more than one water-solubilityenhancing functional group in the protective agent molecule because thewater-solubility is increased to a greater extent. In embodiments wherethe backbone structure is t-butane or phenylmethane one water solubilityenhancing functional group may be sufficient.

In preferred embodiments of this invention, the protective agentcomprises water-solubility enhancing functional groups that are all ofthe same kind, in particular, all of the kind SO₃ ⁻. In alternativeembodiments, the protective agent comprises water-solubility enhancingfunctional groups of different kinds. In as far as the water-solubilityenhancing functional group is SO₃, it is preferred that the protectiveagent comprises at least 2 of these functional groups. Synthesis of theprotective agent can be more efficient and easier, if thewater-solubility enhancing functional groups are all of the same kind.

The backbone structure comprises a moiety selected from the groupconsisting of 9-methylfluorene, t-butane and/or mono-, di- ortriphenylmethane. In preferred embodiments, the backbone structure isselected from 9-methylfluorene and t-butane.

The reactive group is suitable, i.e. has the required chemicalreactivity to undergo a chemical reaction with the functional group tobe protected. It is preferably selected from the group consisting ofoxycarbonyl halogenide, oxycarbonyl O-succinimide, oxycarbonyl Oxymaester, oxycarbonyl anhydride, halogenide, oxymethyl halogenide,hydroxide and thiol groups. When the backbone structure is9-methylfluorene the reactive group is preferably selected fromoxycarbonyl halogenide, oxycarbonyl Oxyma ester and oxycarbonylO-succinimide. When the backbone structure is t-butane the reactivegroup is preferably selected from hydroxide, halogenide, thiol,oxycarbonyl O-succinimide and oxycarbonyl anhydride. When the backbonestructure is selected from mono-, di- and triphenylmethane the reactivegroup is preferably selected from halogenide, oxymethyl halogenide andoxycarbonyl halogenide.

Preferred protective agents having the 9-methylfluorene backbonestructure of the present invention can be illustrated by the followinggeneral formula 1:

wherein R1 to R8 are independently selected from hydrogen, SO₃ ⁻, PO₃²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester and CN with theproviso that at least one, preferably at least two of R1 to R8 isselected from SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ester and CN. In preferred embodiments all of R1 to R8 that are not SO₃⁻, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester or CN arehydrogen.

R99 is preferably selected from the group consisting of oxycarbonylhalogenide, oxycarbonyl O-succinimide, oxycarbonyl Oxyma ester,oxycarbonyl anhydride, halogenide, oxymethyl halogenide, hydroxide andthiol groups, wherein R99 is preferably selected from oxycarbonylhalogenide, oxycarbonyl Oxyma ester and oxycarbonyl O-succinimide.

In preferred embodiments R2 and R7 are selected from SO₃ ⁻, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester and CN, in particular R2and R7 are SO₃ ⁻. Preferably, R1, R3 to R6 and R8 are hydrogen.

In preferred embodiments R3 and R6 are selected from SO₃ ⁻, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester and CN, in particular R3and R6 are SO₃ ⁻. Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

In preferred embodiments R2 and R6 are selected from SO₃ ⁻, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester and CN, in particular R2and R6 are SO₃ ⁻. Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

Preferred protective agents having the 9-methylfluorene backbonestructure of the present invention are shown in the following table 1.The compounds listed in the table are in no way limiting the scope ofthe present invention. They constitute illustrative and preferableprotective agents according to this invention.

TABLE 1 H₂O solubility Pro- Back- enhancing reactive tected # Protectiveagent bone group group group 1

9-methyl- fluorene 2x SO₃ ⁻ oxycarbonyl halogenide amine 2

9-methyl- fluorene 3x SO₃ ⁻ oxycarbonyl halogenide amine 3

9-methyl- fluorene 4x SO₃ ⁻ oxycarbonyl halogenide amine 4

9-methyl- fluorene 2x SO₃ ⁻ Oxyma ester amine 5

9-methyl- fluorene 2x SO₃ ⁻ Oxyma B ester amine 6

9-methyl- fluorene 2x SO₃ ⁻ oxycarbonyl O-succin- imide amine 7

9-methyl- fluorene 2x SO₃ ⁻ oxycarbonyl halogenide amine 8

9-methyl- fluorene 2x N(CH₃)₂ oxycarbonyl halogenide amine 9

9-methyl- fluorene 2x N(CH₃)₃ ⁺ oxycarbonyl halogenide amine 10 

9-methyl- fluorene N(CH₃)₃ ⁺CN oxycarbonyl halogenide amine

Preferred protective agents having the t-butane backbone structure ofthe present invention can be illustrated by the following generalformula 2:

wherein R1 is selected from SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ester, OPO₃ ²⁻ ester and CN. In preferred embodiments R1 is SO₃ ⁻. R99is preferably selected from the group consisting of oxycarbonylhalogenide, oxycarbonyl O-succinimide, oxycarbonyl anhydride,halogenide, oxymethyl halogenide, hydroxide and thiol groups, whereinR99 is more preferably selected from hydroxide, halogenide, thiol,oxycarbonyl O-succinimide and oxycarbonyl anhydride.

Preferred protective agents having the t-butane backbone structure ofthe present invention are shown in the following table 2. The compoundslisted in the table are in no way limiting the scope of the presentinvention. They constitute illustrative and preferable protective agentsaccording to this invention.

TABLE 2 H₂O solubility enhancing reactive protected # Protective agentbackbone group group group 11

t-butane SO₃ ⁻ hydroxide carboxyl 12

t-butane SO₃ ⁻ thiol thiol 13

t-butane SO₃ ⁻ bromide alcohol carboxyl phospono 14

t-butane N(CH₃)₂ bromide alcohol carboxyl phospono 15

t-butane N(CH₃)₃ ⁺ bromide alcohol carboxyl phospono 16

t-butane SO₃ ⁻ Oxy- carbonyl O-succin- imide amine

Preferred protective agents having the mono-, di or triphenylmethanebackbone structure of the present invention can be illustrated by thefollowing general formula 3, 4 or 5, respectively:

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, PO₃²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester and CN with theproviso that at least one, preferably at least two of R1 to R5 areselected from SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ester and CN. In preferred embodiments all of R1 to R5 that are not SO₃⁻, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester or CN arehydrogen.

R99 is preferably selected from the group consisting of oxycarbonylhalogenide, oxycarbonyl Oxyma ester, oxycarbonyl O-succinimide,oxycarbonyl anhydride, halogenide, oxymethyl halogenide, hydroxide andthiol groups, wherein R99 is more preferably selected from halogenide,oxymethyl halogenide and oxycarbonyl halogenide.

In preferred embodiments R3 is selected from SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂,N(CH₃)₃ ⁺, OSO₃ ⁻ ester, OPO₃ ²⁻ ester and CN, in particular R3 is SO₃⁻. Preferably, R1, R2, R4 and R5 are hydrogen.

wherein R1 to R10 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two of R1 to R10 areselected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂,N(CH₃)₃ ⁺ and CN. In preferred embodiments all of R1 to R10 that are notSO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₃ ⁺ or CN arehydrogen.

R99 is preferably selected from the group consisting of oxycarbonylhalogenide, oxycarbonyl Oxyma ester, oxycarbonyl O-succinimide,oxycarbonyl anhydride, halogenide, oxymethyl halogenide, hydroxide andthiol groups, wherein R99 is more preferably selected from halogenide,oxymethyl halogenide and oxycarbonyl halogenide.

In preferred embodiments R3 and R8 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3 and R8 are selected from SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 andR10 are hydrogen.

wherein R1 to R15 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two, most preferably atleast three of R1 to R15 are selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester,OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments allof R1 to R15 that are not SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, OSO₃ ⁻ ester, OPO₃ ²⁻ester, N(CH₃)₃ ⁺ or CN are hydrogen.

R99 is preferably selected from the group consisting of oxycarbonylhalogenide, oxycarbonyl Oxyma ester, oxycarbonyl O-succinimide,oxycarbonyl anhydride, halogenide, oxymethyl halogenide, hydroxide andthiol groups, wherein R99 is more preferably selected from halogenide,oxymethyl halogenide and oxycarbonyl halogenide. In a particularlypreferred embodiment R99 is not halogenide, when R3, R8 and R13 are SO₃⁻. In another particularly preferred embodiment R99 is not chloride,when R3, R8 and R13 are SO₃ ⁻. In a preferred embodiment R99 ispreferably selected from the group consisting of oxycarbonyl halogenide,oxycarbonyl Oxyma ester, oxycarbonyl O-succinimide, oxycarbonylanhydride, oxymethyl halogenide, hydroxide and thiol groups. Inparticularly preferred embodiment R99 is not halogenide, particularlynot chloride.

In preferred embodiments R3, R8 and R13 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3, R8 and R13 are SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 to R12, R14and R15 are hydrogen.

Preferred protective agents having the mono-, di or triphenylmethanebackbone structure of the present invention are shown in the followingtable 3. The compounds listed in the table are in no way limiting thescope of the present invention. They constitute illustrative andpreferable protective agents according to this invention.

TABLE 3 H2O solubility enhancing reactive protected # Protective agentBackbone group group group 17

phenyl- methane SO₃ ⁻ oxycarbonyl- halogenide amine 18

phenyl- methane SO₃ ⁻ halogenide alcohol carboxyl seleno phos- phono 19

phenyl- methane N(CH₃)₂ halogenide alcohol carboxyl seleno phos- phono20

phenyl- methane N(CH₃)₃ ⁺ halogenide alcohol carboxyl seleno phos- phono21

phenyl- methane SO₃ ⁻ oxymethyl- halogenide alcohol 22

diphenyl- methane 2x SO₃ ⁻ oxycarbonyl- halogenide amine 23

triphenyl- methane 3x SO₃ ⁻ halogenide alcohol

Preferably, the functional group is present on an amino acid, peptide orprotein and the chemical reaction is peptide or protein synthesis in asolvent as mentioned above and below.

The protective agents as mentioned above and below provide a protectinggroup to the amino acids or peptides. In preferred embodiments, theα-amine protecting group is at least one of the following formulas 6 to10. In each case the nitrogen shown in the following general formulabelongs to the protected amino group:

wherein R1 to R15 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two, most preferably atleast three of R1 to R15 are selected from SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, OSO₃⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₃ ⁺ and CN. In preferred embodiments allof R1 to R15 that are not SO₃ ⁻, PO₃ ²⁻, N(CH₃)₂, OSO₃ ⁻ ester, OPO₃ ²⁻ester, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3, R8 and R13 are selected from SO₃ ⁻, PO₃ ²⁻,OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3, R8 and R13 are SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 to R12, R14and R15 are hydrogen.

wherein R1 to R8 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R8 is selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN. Inpreferred embodiments all of R1 to R8 that are not SO₃ ⁻, PO₃ ² OSO₃ ⁻ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R2 and R7 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR2 and R7 are SO₃ ⁻. Preferably, R1, R3 to R6 and R8 are hydrogen.

In preferred embodiments R3 and R6 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3 and R6 are SO₃ ⁻. Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

In preferred embodiments R2 and R6 are selected from SO₃ ⁻, PO₃ ²⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R2 andR6 are SO₃ ⁻. Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, PO₃ ²⁻,N(CH₃)₂, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃⁻. Preferably, R1, R2, R4 and R5 are hydrogen.

wherein R1 to R10 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two of R1 to R10 areselected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂,N(CH₃)₃ ⁺ and CN. In preferred embodiments all of R1 to R10 that are notSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ or CN arehydrogen.

In preferred embodiments R3 and R8 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3 and R8 are selected from SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 andR10 are hydrogen.

wherein R1 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments R1 is SO₃ ⁻.

In particularly preferred embodiments the α-amine protecting group is a9-(2-sulfo)fluorenylmethyloxycarbonyl group (Sulfmoc),9-(3,6-disulfo)fluorenylmethyloxy-carbonyl group,9-(2,7-disulfo)fluorenylmethyloxy-carbonyl group or atert-butyl-(2-sulfonate)oxycarbonyl group (Sboc). As used herein, theterm “Smoc” denotes a 9-(3,6-disulfo)fluorenylmethyloxy-carbonyl groupor a 9-(2,7-disulfo)fluorenylmethyloxy-carbonyl group.

In a preferred embodiment of the invention, any reactive side chainfunctional group of said first amino acid or first peptide and/or saidsecond amino acid or second peptide is protected with a side chainprotecting group, which preferably comprises at least one watersolubility enhancing functional group. The side chain protecting groupmay comprise a sulfonic group or a sulfonic ester. The method preferablycomprises the step of removing the side chain protecting groups. Thewater solubility enhancing functional group of the side chain protectinggroup provides solubility to the side chain protected amino acid orpeptide. Suitable side chain protecting groups are the reaction productsof one of the protective agents mentioned above with the respective sidechain functional group of the amino acid or peptide.

Regarding the side chain protecting groups, preferred amine protectinggroups are those shown in the following formulae 11 to 15, wherein thenitrogen belongs to the protected amino group.

wherein R1 to R15 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two, most preferably atleast three of R1 to R15 are selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments allof R1 to R15 that are not SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester,N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3, R8 and R13 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3, R8 and R13 are SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 to R12, R14and R15 are hydrogen.

wherein R1 to R8 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R8 is selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R8 that are not SO₃ ⁻, PO₃ ²⁻,OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R2 and R7 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR2 and R7 are SO₃ ⁻. Preferably, R1, R3 to R6 and R8 are hydrogen.

In preferred embodiments R3 and R6 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3 and R6 are SO₃ ⁻. Preferably, R1, R2, R4, R5, R7 and R8 are hydrogen.

In preferred embodiments R2 and R6 are selected from SO₃ ⁻, PO₃ ²⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R2 andR6 are SO₃ ⁻. Preferably, R1, R3, R4, R5, R7 and R8 are hydrogen.

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, PO₃ ²⁻,OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃⁻. Preferably, R1, R2, R4 and R5 are hydrogen.

wherein R1 to R10 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two of R1 to R10 areselected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂,N(CH₃)₃ ⁺ and CN. In preferred embodiments all of R1 to R10 that are notSO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ or CN arehydrogen.

In preferred embodiments R3 and R8 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3 and R8 are selected from SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 andR10 are hydrogen.

wherein R1 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments R1 is SO₃ ⁻.

In particularly preferred embodiments, the side chain amine protectinggroup is selected from 9-(2-sulfo)fluorenylmethyloxycarbonyl group(Sulfmoc), 9-(3,6-disulfo)fluorenylmethyloxy-carbonyl group,9-(2,7-disulfo)fluorenylmethyloxy-carbonyl group,tri(4-sulfophenyl)methyl group (SulfoTrt),tert-butyl-(2-sulfonate)oxycarbonyl group (Sboc) and4-sulfo-carbobenzyloxy group (SulfoCBz).

Preferred alcohol protecting groups are shown in the following formulae16 to 19. In each case the oxygen shown in the following general formulabelongs to the protected alcohol group:

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, PO₃ ²⁻,OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃⁻. Preferably, R1, R2, R4 and R5 are hydrogen.

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃⁻. Preferably, R1, R2, R4 and R5 are hydrogen.

wherein R1 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments R1 is SO₃ ⁻.

wherein R1 to R15 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two, most preferably atleast three of R1 to R15 are selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester,OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments allof R1 to R15 that are not SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester,N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3, R8 and R13 are selected from SO₃ ⁻, PO₃ ²⁻,OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3, R8 and R13 are SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 to R12, R14and R15 are hydrogen.

In particularly preferred embodiments the alcohol protecting groups areselected from 4-sulfobenzyl group (BzS), 4-sulfo-benzyloxymethyl group(BOMS), tri(4-sulfophenyl)methyl group (SulfoTrt) andtert-butyl-1-sulfonate group (tBuS).

Preferred thiol protecting groups are shown in the following formulae 20and 21. In each case the sulfur shown in the following general formulabelongs to the protected thiol group:

wherein R1 to R15 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two, most preferably atleast three of R1 to R15 are selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester,OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments allof R1 to R15 that are not SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester,N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3, R8 and R13 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3, R8 and R13 are SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 to R12, R14and R15 are hydrogen.

wherein R1 is selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester,N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments R1 is SO₃ ⁻.

In particularly preferred embodiments the thiol protecting group isselected from tri(4-sulfophenyl)methyl group (SulfoTrt) and1-sulfo-2-methyl-2-propanethiol group (StBuS).

Preferred carboxyl protecting groups are those shown in the followingformulae 22 to 25. In each case the oxygen shown in the followinggeneral formula belongs to the protected carboxyl group:

wherein R1 to R15 are independently selected from hydrogen, SO₃ ⁻, OSO₃⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with theproviso that at least one, preferably at least two, most preferably atleast three of R1 to R15 are selected from SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester,OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments allof R1 to R15 that are not SO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester,N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3, R8 and R13 are selected from SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particularR3, R8 and R13 are SO₃ ⁻. Preferably, R1, R2, R4 to R7, R9 to R12, R14and R15 are hydrogen.

wherein R1 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments R1 is SO₃ ⁻.

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃ ⁻.Preferably, R1, R2, R4 and R5 are hydrogen.

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, PO₃ ²⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃ ⁻.Preferably, R1, R2, R4 and R5 are hydrogen.

In particularly preferred embodiments the carboxyl protecting groups areselected from 4-sulfobenzyl group (BzS), 4-sulfo-benzyloxymethyl group(BOMS), tri(4-sulfophenyl)methyl (SulfoTrt) group andtert-butyl-1-sulfonate group (tBuS).

Preferred seleno protecting groups are those shown in the followingformula 26. In each case the selenium shown in the following generalformula belongs to the protected seleno group:

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃ ⁻.Preferably, R1, R2, R4 and R5 are hydrogen.

Preferred phosphono protecting groups are those shown in the followingformulae 27 and 28. In each case the oxygen shown in the followinggeneral formula belongs to the protected phosphono group:

wherein R1 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺ and CN. In preferred embodiments R1 is SO₃ ⁻.

wherein R1 to R5 are independently selected from hydrogen, SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN with the provisothat at least one, preferably at least two of R1 to R5 are selected fromSO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN.In preferred embodiments all of R1 to R5 that are not SO₃ ⁻, OSO₃ ⁻ester, OPO₃ ²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ or CN are hydrogen.

In preferred embodiments R3 is selected from SO₃ ⁻, OSO₃ ⁻ ester, OPO₃²⁻ ester, PO₃ ²⁻, N(CH₃)₂, N(CH₃)₃ ⁺ and CN, in particular R3 is SO₃ ⁻.Preferably, R1, R2, R4 and R5 are hydrogen.

The following is an overview over the most preferred protecting groups.

Amine Protecting Groups

Alcohol Protecting Groups

Thiol Protecting Groups

Carboxyl Protecting Groups

Phosphono Protecting Groups

Preferably, the amino protecting groups mentioned above are used toprotect the main chain amino group and to provide an anchoring point tothe amino acid or peptide to the solid phase, if an ion exchanger isused.

In a specific embodiment, said protecting group having awater-solubility enhancing group preferably comprises at least twowater-solubility enhancing groups, preferably ionic groups. Thisembodiment provides the advantage that a higher bonding to an ionexchanger can be achieved.

In another embodiment, said protecting group having a water-solubilityenhancing group preferably comprises exactly one water-solubilityenhancing group, preferably ionic group. This embodiment provides theadvantage that a lower hydrolyzation of protecting groups can beachieved such that a reaction in water or other protic solvent provideshigher yield.

Preferably, said protecting group having a water-solubility enhancinggroup comprises a fluorenylmethoxycarbonyl residue (Fmoc) being modifiedwith a water-solubility enhancing group, preferably ionic group.

According to an embodiment of the invention, it is preferably providedthat any reactive side chain functional group of said amino acid orpeptide, preferably first amino acid or first peptide and/or said secondamino acid or second peptide, is protected with a side chain protectinggroup. Astonishing improvements regarding to solubility of the firstamino acid or first peptide and/or said second amino acid or secondpeptide can be achieved by that embodiment. In addition thereto, thebinding of first amino acid or first peptide to an ion exchanger ispreferably improved.

In a specific embodiment, said side chain protecting group comprises atleast one water solubility enhancing functional group, preferably ionicgroup.

In another embodiment, said side chain protecting group does notcomprise any water solubility enhancing functional group.

In methods according to the invention, it can also be provided that thefunctional group of said first amino acid or first peptide and saidsecond amino acid or second peptide being protected is selected fromα-amino, side chain amino, thiol, carboxyl, hydroxyl, phosphono andseleno.

In a preferred embodiment of the present invention the second amino acidor second peptide does not comprise preferably any protecting groupswith the exception of groups protecting a primary amine group and withthe exception of groups protecting a carboxylic group. More preferably,the second amino acid or second peptide does not preferably comprise anyprotecting groups with the exception of groups protecting a primaryamine group. This embodiment provides astonishing cost improvements andan easier processing. With regard to achieve a higher solubility of theamino acid being coupled to a growing peptide chain a protecting groupmight be used in some cases.

Furthermore, the solubility of the second amino acid or second peptideis preferably improved by the use of a salt of the second amino acid orsecond peptide and the salt comprises a soluble organic ion such asquaternary amino cations. Furthermore, compounds imparting solubility tosalts in organic solvents are preferably applied, such as crown ethers.Additionally, the second amino acid or second peptide can be added tothe reaction mixture in solid phase and a transport of the added secondamino acid or second peptide to the first amino acid or first peptide isachieved via the solvent. This embodiment comprises cost advantages.However, the reaction time is longer than in other embodiments.

The solubility of the second amino acid or second peptide is preferablysufficient for achieving an adequate concentration of the second aminoacid or second peptide in the mixture added to the activated first aminoacid or first peptide and/or the growing chain being obtained. Asmentioned above, the solubility of the second amino acid or secondpeptide can be improved if useful. However, in many cases there is noneed to improve the solubility of the second amino acid or secondpeptide.

Preferably, the following amino acids are used without any protectiongroup as the second amino acid or second peptide Alanine (Ala), Glycine(Gly), Isoleucine (Ile), Leucine (Leu), Proline (Pro), Valine (Val),Phenylalanine (Phe), Arginine (Arg), Asparagine (Asn), Glutamine (Gin),Tyrosine (Tyr), Tryptophan (Trp), Histidine (His), Methionine (Met),more preferably Ala, Gly, Ile, Leu, Pro, Val, Phe. These amino acidshave conventionally a sufficient solubility for achieving an adequateconcentration of the second amino acid or second peptide in the mixtureadded to the activated first amino acid or first peptide and/or thegrowing chain being obtained.

The amino acids Glutamic acid (Glu), Aspartic acid (Asp), Cysteine(Cys), Lysine (Lys), Serine (Ser), Threonine (Thr) comprise preferably aprotection group, more preferably a side chain protection group asmentioned above and below.

Preferably, said amino acid or peptide comprising a protecting grouphaving a water-solubility enhancing group is added to said ion exchangerin an amount of at least 10%, more preferably at least 80% of thecapacity of the ion exchanger.

Preferably, said amino acid or peptide being bound to a solid phase isadded to said solid phase in an amount of at least 10%, more preferablyat least 80% of the capacity of the solid phase.

According to the invention, it is preferably provided that said formingof the peptide bond is achieved using a solution of second amino acid orpeptide wherein the concentration of the second amino acid or the secondpeptide is in the range of 0.01 to 90% by weight, preferably in therange of 0.1 to 50% by weight, more preferably in the range of 0.5 to20% by weight based on the total weight of the solution.

In an embodiment of the present invention, forming of the peptide bondis preferably achieved at a temperature in the range of −20 to 100° C.,more preferably 0 to 50° C., even more preferably 10 to 30° C.

In a further embodiment of the present invention, forming of the peptidebond is preferably achieved at a pH value in the range of 4 to 12,preferably in the range of 6 to 10, more preferably in the range of 7 to9.5, even more preferably in the range of 7 to 9.0 and even morepreferably in the range of 7 to 8.5.

According to the invention, it is preferably provided that the peptidebeing obtained by forming of the peptide bond comprises 2 to 150 aminoacid units, more preferably 2 to 6 amino acid units, even morepreferably 2 to 4 amino acid units.

According to a further embodiment of the present invention, it ispreferably provided that the peptide being obtained by forming of thepeptide bond comprises 4 to 300 amino acid units, more preferably 10 to100 amino acid units, even more preferably 15 to 45 amino acid units.Peptides having a high number of amino acid units are preferablyachieved by a method wherein a first amino acid or first peptide iscovalently bound to a solid phase.

As mentioned above and below some of the embodiments of the presentinvention include a deprotection step. Conventionally, a deprotectionsolution is applied in order to achieve a deprotection of the peptide ora specific amino acid unit of the peptide.

The deprotecting solutions that are suitable for use in the presentinvention preferably comprise an acid or base, preferably an aqueousacid or base, i.e. the acid and/or base preferably is water-soluble.Preferred acids are phosphoric acid, hydrochloric acid or trifluoroacetic acid. Preferred bases are amines and ammonia. Preferreddeprotecting solutions are amine and/or ammonia solutions. The base isused in an amount and to the extent necessary to deprotect thefunctional group. The solubility of certain organic bases may limit theamount that can be dissolved in the water, alcohol or mixture of waterand alcohol. Suitable bases are those having a solubility that allowsfor dissolving a sufficient amount to carry out the deprotection in theselected solvent.

The deprotecting solution for deprotecting a Sboc group preferablycomprises an aqueous acid, such as phosphoric acid, hydrochloric acid ortrifluoro acetic acid.

The deprotecting solution for deprotecting a Smoc group, a Sulfmoc groupand/or similar groups such as fluorenylmethyloxy-carbonyl groups beingderived from compounds or groups of formulae 1, 7, 12 etc. preferablycomprises a water soluble base such as amine, ammonia or inorganichydroxides. The water soluble base is used in an amount and to theextent necessary to deprotect the peptide. The solubility of certainorganic bases may limit the amount that can be dissolved in the water,alcohol or mixture of water and alcohol. Suitable bases are those havinga solubility that allows for dissolving a sufficient amount to carry outthe deprotection in the selected solvent.

Surprising improvements for deprotecting a Smoc group, a Sulfmoc groupand/or similar groups can be achieved by using at least 5% of an organicbase, such as primary, secondary and/or tertiary amines, preferably,piperazine, piperidine, ethanolamine and/or ethylendiamine in water ormixtures of water and environmentally friendly solvents including proticsolvents, such as water, primary, secondary and/or tertiary alcohols,non-protic solvents such as ketones, nitriles, lactones, lactams, carbonacid amides, carbon acid ester, ether, urea derivatives, sulfoxides,sulfones, carbonate ester. Preferred examples of non-protic solvents aree. g. dimethylacetamide, ethyl methyl ketone, acetone, methyl isopropylketone, methyl isobutyl ketone, methyl n-butyl ketone, methyl t-butylketone, methyl isoamyl ketone, dimethyl sulfoxide, hexamethylphosphorictriamide, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol,dimethyl ether, ethyl acetate, tertiary butyl acetate, or mixture(s)thereof. This reduces base catalysed side reactions (e.g. thedeprotection of OtBu based side chain protecting groups, cleavage ofesters with SPPS linker molecules) during deprotection of the Smocgroup, the Sulfmoc group and/or similar groups compared to the usage ofammonia or inorganic hydroxides. This results in a higher purity andproduct yield.

It will be understood that after deprotection, the reaction anddeprotection steps can be repeated using further amino acids or furtherpeptides wherein the α-amino group or the carboxylic group of saidfurther amino acid and/or further peptide is preferably protected with aprotecting group according to this invention, in particular an Smocgroup or an Sboc group, until the desired target peptide is obtained.

In an embodiment, the method further comprises the step of releasing theresulting peptide from the polymeric support with a releasingcomposition when the peptide is complete.

In the case that the peptide is ionically bound to an ion exchanger by aprotecting group having a water-solubility enhancing group, thereleasing composition is preferably a composition deprotecting thepeptide from the corresponding protecting group or a composition beingable to release the peptide from the ion exchanger and maintaining theprotection of the peptide. The deprotecting agent being used forreleasing the peptide from the solid support depends on the protectinggroup being used. A Sboc group is conventionally released by an acidiccomposition while basic reagents are used to remove the Smoc moiety.

In a specific embodiment of the present invention, the forming a peptidebond is preferably achieved while an amino acid or a peptide isionically bound to an ion exchanger and after forming the peptide bondthe obtained peptide is removed from the ion exchanger by deprotectingthe obtained peptide. In preferred embodiments at least one preferablyat least 50% and more preferably at least 90% of side chain protectinggroups that are attached to the amino acids in the target peptide areremoved by the releasing composition.

In another embodiment of the present invention, the forming a peptidebond is preferably achieved while an amino acid or a peptide isionically bound to an ion exchanger and after forming the peptide bondthe obtained peptide is removed from the ion exchanger by adding a ionicsolution and maintaining the protection of the obtained peptide.

Preferably, release is carried out in the presence of scavengercompositions (e.g. water, phenol, DTT, triethylsilane and anisole),which protect the peptide from undesired side reactions during and afterthe cleaving step. A skilled person can select a suitable scavenger withregard to the protecting groups that are present.

The released peptide can be separated from the ion exchanger byfiltration and the peptide can then be recovered from the filtrate by aconventional step such as evaporation or solvent-driven precipitation.

In the case that the peptide is covalently bound to a solid support, anappropriate cleaving composition is preferably used. Suitable cleavingcompositions and methods are well known to a skilled person. Typically,an acid, such as trifluoroacetic acid and hydrofluoric acid (HF), isused to carry out the cleaving step. Preferably, an acid suitable forcleaving the desired peptide from the polymeric support concurrentlyremoves side chain protecting groups that are attached to the aminoacids in the target peptide.

Preferably, cleavage or release is carried out in the presence ofscavenger compositions (e.g. water, phenol, DTT, triethylsilane andanisole), which protect the peptide from undesired side reactions duringand after the cleaving step. A skilled person can select a suitablescavenger with regard to the protecting groups that are present.

The cleaved or released peptide can be separated from the cleavedsupport (e.g. a resin) by filtration and the peptide can then berecovered from the filtrate by a conventional step such as evaporationor solvent-driven precipitation.

In another preferred embodiment of the invention, the method furthercomprises at least one washing step using a solvent comprising a proticand/or non-protic solvent as mentioned above and below regarding theforming of the peptide bond, and a step of collecting waste solutionsobtained in the washing steps, contacting the waste solutions to anaffinity chromatography column, preferably a solid anion and/or cationexchange support, thereby retaining waste compounds that comprise awater-solubility enhancing functional group, particular a chargedfunctional group like SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, PO₃ ²⁻, andN(CH₃)₃ ⁺. It has been particularly effective to use an anion exchangesupport for removal of compounds that contain at least one sulfonicgroup. Subsequently, the waste compounds may be collected byregeneration of the affinity chromatography column, preferably a solidanion and/or cation exchange support, and disposed. Alternatively, theaffinity chromatography column, preferably an anion and/or cationexchange support material, containing the waste compounds may bedisposed.

Washing steps are typically carried out after the coupling step andafter the deprotection step. The anion and/or cation exchange stepallows for the removal of all sulfo-containing compounds, such asunreacted or cleaved protecting groups and reagents, e.g. couplingagents and capping reagents as described herein from the washingsolutions. The waste compounds are retained on the anion or cationexchange column and a purified solvent is obtained. The retainedcompounds can be disposed together with the anion or cation exchangematerial or after elution and only a minimum amount of chemical wastehas to be disposed.

In a further preferred embodiment of the invention, the method furthercomprises a purification step after the releasing step wherein thesolution containing the deprotected and released target protein orpeptide is contacted to an affinity chromatography column, preferably asolid anion and/or cation exchange support, thereby retaining wastecompounds such as those comprising a sulfonic group, and collecting thepurified target protein. In preferred embodiments the waste compoundscomprise at least one sulfonic group as part of the protecting groupsand other reagents. Due to the sulfo-containing protecting groups, andthe sulfo-containing reagents it is possible to remove substantially allof the excess chemicals and side products generated during the coupling,the deprotection and the cleaving reactions in a protic and/ornon-protic solvent as mentioned above and below by affinitychromatography column, preferably ion exchange methods. Of course, thisalso works with the other charged functional groups. Only the targetpeptide is able to run through the ion exchange column whereas thepeptide side products, protecting group residues and excess reagents areretained on the column. After regeneration of the column, only a minimumamount of chemical waste has to be disposed. This is a beneficial effectof using charged functional groups such as SO₃ ⁻, OSO₃ ⁻ ester, OPO₃ ²⁻ester, PO₃ ²⁻, and N(CH₃)₃ ⁺. But even if one or more of the unchargedfunctional groups are used, the process can still be carried out inwater or in another environmentally friendly solvent, thereby reducingthe amount of waste chemicals compared to prior art processes.

A further subject matter of the present invention is a peptideobtainable by a method according to the present invention.

A further subject matter of the present invention is an amino acid or apeptide comprising a protecting group having a water-solubilityenhancing group being bound to the amino group and an activated or freecarboxyl group, preferably an activated carboxyl group. Preferably, thepeptide is ionically bound to an ion exchanger.

It is preferably provided that the amino acid or peptide according tothe present invention preferably has 1 to 150 amino acid units,preferably 1 to 50 amino acid units, more preferably 1 or 2 amino acidunits.

Another aspect of the present invention relates to modified amino acids,peptides and salts thereof comprising a protecting group selected fromthe group consisting of those shown above under formulae 6 to 25, morepreferably selected from those shown above under formulae 6 to 10. Inparticularly preferred embodiments these protecting groups include9-(2-sulfo)fluorenylmethyloxycarbonyl group (Sulfmoc),9-(3,6-disulfo)fluorenylmethyloxy-carbonyl group,9-(2,7-disulfo)fluorenylmethyloxy-carbonyl group,tri(4-sulfophenyl)methyl group (SulfoTrt),tert-butyl-(2-sulfonate)oxycarbonyl group (Sboc), 4-sulfo-carbobenzyloxygroup (SulfoCBz), tert-butyl-1-sulfonate group (tBuS),1-sulfo-2-methyl-2-propanethiol group (StBuS), 4-sulfobenzyl group(BzS), and 4-sulfo-benzyloxymethyl group (BOMS).

The invention is illustrated but not limited by the following examples.

EXAMPLES Example 1: Synthesis of9-(3,6-disulfo)fluorenylmethyloxycarbonyl chloride (Smoc-Cl)

2 g (7.73 mmol) of Fmoc-chloride was treated with 20 mL of concentratedsulfuric acid. After work up of the reaction mixture 2.96 g (7.07 mmol,91.4%) of crude Smoc-chloride was obtained in form of a slightly yellowsolid.

Analytical Data of Smoc-Chloride:

¹H NMR (500 MHz, D₂O) δ=7.80 (s, 2H), 7.69 (d, J=7.9 Hz, 2H), 7.48 (d,J=7.8 Hz, 2H), 3.84 (d, J=4.8 Hz, 2H), 3.45 (t, J=4.7 Hz, 1H).

¹³C NMR (126 MHz, D₂O) δ=145.57, 142.54, 141.65, 125.16, 121.61, 120.90,62.54, 49.65.

Example 2: Synthesis of 9-(2,7-disulfo)fluorenylmethyloxycarbonylchloride (Smoc-Cl)

2 g (7.73 mmol) of Fmoc-chloride were treated with 20 mL of concentratedsulfuric acid and heated to 100° C. Sulfuric acid was neutralised withNaOH (pH9.5) and solvent removed under reduced pressure and NMRanalytics confirmed formation of target intermediate. The intermediatewas dissolved again in 20% sulfuric acid in water, stirred for 6 h toform 9-(2,7-disulfo)fluorenylmethanol. Sulfuric acid was neutralisedwith NaOH (pH 6.7) and the solvent removed under reduced pressure. Asolution of 1.2 eq. phosgene in 25 ml of DCM was cooled to 0° C. and9-(2,7-disulfo)fluorenylm ethanol was added slowly under stirring(Carpino and Han, The Journal of Organic Chemistry 1972, 37, (22),3404-3409). The solution was stirred for 1 h in the ice bath and thenlet stand for 4 h at ice-bath temperature. Solvent and excess phosgenewere removed under reduced pressure giving the corresponding product.

NMR Intermediate:

¹H NMR (300 MHz, D₂O) δ: 6.09 (s, 2H), 7.23-7.40 (m, 2H), 7.72 (s, 2H),7.95 (d, J=6.2 Hz, 2H).

¹³C NMR (75 MHz, D₂O) δ: 142.61, 132.99, 131.74, 130.23, 129.28, 127.22,125.57, 124.69.

LC-APCI-MS for 9-(2,7-disulfo)-fluorenylmethyloxycarbonyl chloride:LC-APCI-MS calculated for C₁₅H₉ClO₂₂. m/z: 256.03. Measured m/z: 256.94[M-H-2×SO₃]⁻.

Example 3: Synthesis of Smoc-ß Ala-OH (Smoc-ß-alanine)

8.41 mmol of Fmoc-ß-alanine were treated with 30 mL of concentratedsulfuric acid. After work up of the reaction mixture 8.09 mmol (96.2%)of crude Smoc-ß-alanine were obtained.

Example 4: Synthesis of L-carnosine

Smoc-ß-alanine is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-ß-alanine is activated by 3equivalents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2equivalents N-hydroxysuccinimide (NHS) in water.

Thereafter, the ion exchanger loaded with the activated Smoc-ß-alanineis washed two times with water. After the washing steps, 1.5 equivalentsL-histidine solved in water are added to the activated Smoc-ß-alanineand reacted at 4° C. for 12 minutes at pH 7.5 under agitation. Excess ofL-histidine and released NHS are removed by washing with water.

The formed L-carnosine is released by deprotection using 0.25 mM NaOH(pH 10, 20 minutes) or Na₂CO₃ (pH 10, 20 minutes).

Example 5: Synthesis of L-carnosine

Smoc-ß-alanine is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-proline is obtained in a similarmanner as disclosed above with regard to Smoc-ß-alanine (see Example 3).Smoc-ß-alanine is activated by 3 equivalent1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)4:1 ratio (volume). The activation was much higher than in example 4.

Thereafter, the ion exchanger loaded with the activated Smoc-ß-alanineis washed two times with water. After the washing step, 1.5 equivalentsL-histidine solved in water are added to the activated Smoc-ß-alanineand reacted at 4° C. for 12 minutes at pH 7.5 under agitation. Excess ofL-histidine and released NHS are removed by washing with water.

The yield in example 5 is higher than the yield in example 4.

The formed L-carnosine is released by deprotection using 0.25 M NaOH(pH10, 10 minutes) or Na₂CO₃ (pH 10, 20 minutes).

Example 6: Synthesis of Pro-Tyr-OMe

Smoc-proline is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-proline is activated by 3equivalents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2equivalents N-hydroxysuccinimide (NHS) in mixture of water andtetrahydrofuran (THF) 3:1 ratio (volume).

Thereafter the ion exchanger loaded with the activated Smoc-proline iswashed two times with water. After the washing step, 1.5 equivalentsL-tyrosine methyl ester solved in water are added to the activatedSmoc-proline and reacted at 24° C. for 12 minutes at pH 8 underagitation. Excess of L-tyrosine methyl ester and released NHS areremoved by washing with water.

The formed Pro-Tyr-OMe is released by deprotection using 0.5 M NaOH(pH9, 25 minutes) or Na₂CO₃ (pH 10, 20 minutes).

Example 7: Synthesis of Pro-Tyr-OMe

Smoc-proline is added to an ion exchange resin (Amberlite IRA-900; SigmaAldrich) at a maximum load. Smoc-proline is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and tetrahydrofuran (THF)3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-proline iswashed twice with water. After the washing step, 1.5 equivalentsL-tyrosine methyl ester solved in water are added to the activatedSmoc-proline and reacted at 24° C. for 12 minutes at pH 8 underagitation. Excess of L-tyrosine methyl ester and released NHS areremoved by washing with water.

The yield in Example 7 is lower than the yield in Example 6.

The formed Pro-Tyr-OMe is released by deprotection using 0.5 mM NaOH(pH10, 15 minutes) or Na₂CO₃ (pH 10, 20 minutes).

Example 8: Synthesis of Pro-Tyr-OMe

Smoc-proline is added to an ion exchange resin (Amberlite IRA-96; SigmaAldrich) at a maximum load. Smoc-proline is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and tetrahydrofuran (THF)4:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-proline iswashed thrice with water. After the washing steps, 2 equivalentsL-tyrosine methyl ester solved in water are added to the activatedSmoc-proline and reacted at 15° C. for 25 minutes at pH 8.2 underagitation. Excess of L-tyrosine methyl ester and released NHS areremoved by washing with water.

The yield in Example 8 is similar to the yield in Example 7.

The formed Pro-Tyr-OMe is released by deprotection using 0.25M CaOH₂(pH10, 15 minutes) or CaCO₃ (pH 10, 30 minutes).

Example 9: Synthesis of Pro-Tyr-OMe

Smoc-proline is added to an ion exchange resin (Amberlite IRA-410; SigmaAldrich) at a maximum load. Smoc-proline is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and tetrahydrofuran (THF)4:1 ratio (volume).

Thereafter. the ion exchanger loaded with the activated Smoc-proline iswashed thrice with water. After the washing steps, 2 equivalentsL-tyrosine methyl ester solved in water are added to the activatedSmoc-proline and reacted at 15° C. for 25 minutes at pH 8.2 underagitation. Excess of L-tyrosine methyl ester and released NHS areremoved by washing with water.

The yield in Example 9 is lower than the yield in Example 6.

The formed Pro-Tyr-OMe is released by deprotection using 1M NaOH (pH10,10 minutes) or Na₂CO₃ (pH 10, 20 minutes).

Example 10: Synthesis of Pro-Tyr-OMe

Smoc-Proline is added to an ion exchange resin (Amberlite IRA-958; SigmaAldrich) at a maximum load. Smoc-Proline is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-Hydroxysuccinimide (NHS) in mixture of water and tetrahydrofuran (THF)4:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-proline iswashed thrice with water. After the washing steps, 2 equivalentsL-tyrosine methyl ester solved in water are added to the activatedSmoc-proline and reacted at 15° C. for 25 minutes at pH 8.2 underagitation. Excess of L-tyrosine methyl ester and released NHS areremoved by washing with water.

The yield in Example 10 is higher than the yield in Example 10.

The formed Pro-Tyr-OMe is released by deprotection using 25% NH₃(aq)(pH10, 20 minutes) or (NH₄)₂CO₃ (pH 10, 20 minutes).

Example 11: Synthesis of L-carnosine

Smoc-ß-alanine is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-ß-alanine is activated by2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) in mixture ofwater and isopropyl alcohol 3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-ß-alanineis washed one time with water. After the washing step, 1.5 equivalentL-histidine solved in water are added to the activated Smoc-ß-alanineand reacted at 24° C. for 15 minutes at pH 7.8 under agitation. Excessof L-histidine and released NHS are removed by washing with water.

The yield in Example 11 is similar to the yield in Example 4.

The formed L-carnosine is released by deprotection using 0.5 M KOH(pH9.8, 12 minutes) or K₂CO₃ (pH 10, 20 minutes).

Example 12: Synthesis of Smoc-Gly-His-OH

Smoc-glycine is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-glycine is obtained in a similarmanner as disclosed above with regard to Smoc-ß-alanine (see Example 3).Smoc-glycine is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and tetrahydrofuran (THF)3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-glycine iswashed thrice with water. After the washing steps, 1.5 equivalentsL-histidine solved in water are added to the activated Smoc-glycine andreacted at 18° C. for 20 minutes at pH 8.5 under agitation. Excess ofL-histidine and released NHS are removed by washing with water.

The formed Smoc-Gly-His-OH is released by eluting with 1M NaCl

Solution Example 13: Synthesis of Asn-Asn-OH

Smoc-Asn is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Asn is obtained in a similar manneras disclosed above with regard to Smoc-ß-alanine (see Example 3).Smoc-Asn is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and and isopropyl alcohol4:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Asn iswashed three times with water. After the washing step, 1.5 equivalentL-Asn solved in water are added to the activated Smoc-Asn and reacted at8° C. for 25 minutes at pH 8.7 under agitation. Excess of L-Asn andreleased NHS are removed by washing with water.

The formed Asn-Asn-OH is released by deprotection using 0.5M NaOH(pH9.5, 12 minutes) or K₂CO₃ (pH 10, 25 minutes).

Example 14: Synthesis of Asn-Gln-OH

Smoc-Asn is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Asn is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentN-hydroxysuccinimide (NHS) in mixture of water and isopropyl alcohol 4:1ratio (volume).

Thereafter the ion exchanger loaded with the activated Smoc-Asn iswashed twice with water. After the washing steps, 1.5 equivalents L-Glnsolved in water are added to the activated Smoc-Asn and reacted at 24°C. for 20 minutes at pH 9 under agitation. Excess of L-Gln and releasedNHS are removed by washing with water.

The formed Asn-Gln-OH is released by deprotection using 1M NaOH (pH9.5,12 minutes) or K₂CO₃ (pH 10, 25 minutes).

Example 15: Synthesis of Asp(OtBu)-Glu-OH

Smoc-Asp(OtBu)-OH is added to an ion exchange resin (DEAE Sephadex A-25;GE Healthcare) at a maximum load. Smoc-Asp(OtBu)-OH is obtained in asimilar manner as disclosed above with regard to Smoc-ß-alanine (seeExample 3). Smoc-Asp(OtBu)-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and2-methyltetrahydrofuran 4:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Asp(OtBu)is washed twice with water. After the washing steps, 1.5 equivalentsL-Glu solved in water are added to the activated Smoc-Asp(OtBu) andreacted at 24° C. for 20 minutes at pH 9 under agitation. Excess ofL-Glu and released NHS are removed by washing with water.

The formed Asp(OtBu)-Glu-OH is released by deprotection using 1M NaOH(pH9.5, 12 minutes) or K₂CO₃ (pH 10, 25 minutes).

Example 16: Synthesis of Ile-D-Val-OH

Smoc-Ile-OH is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Ile-OH is obtained in a similarmanner as disclosed above with regard to Smoc-ß-alanine (see Example 3).Smoc-Ile-OH is activated by 4 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 3 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and dimethyl sulfoxide(DMSO) 3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Ile iswashed twice with water. After the washing steps, 1.5 equivalents D-Valsolved in water are added to the activated Smoc-Ile and reacted at 25°C. for 17 minutes at pH 8.5 under agitation. Excess of D-Val andreleased NHS are removed by washing with water.

The formed Ile-D-Val-OH is released by deprotection using 1M NaOH (pH9.5, 12 minutes) or Na₂CO₃ (pH 10, 25 minutes).

Example 17: Synthesis of Sulfmoc-Gly-Val-OH

9-(2-Sulfo)fluorenylmethyloxycarbonylglycine (Sulfmoc-Gly-OH) isobtained in a similar manner as disclosed above with regard toSmoc-ß-alanine (see Example 3).9-(2-Sulfo)fluorenylmethyloxycarbonylglycine (Sulfmoc-Gly-OH) isactivated by 3 equivalents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid(EDC) and 2 equivalents N-hydroxysuccinimide (NHS) in water. Afterwards,the preactivated Sulfmoc-Gly is loaded to an ion exchange resin (DEAESephadex A-25; GE Healthcare).

Thereafter, the ion exchanger loaded with the activated Sulfmoc-Gly iswashed twice with water. After the washing steps, 1.5 equivalents L-Valsolved in water are added to the activated Sulfmoc-Gly and reacted at25° C. for 30 minutes at pH 9 under agitation. Excess of L-Val andreleased NHS are removed by washing with water.

The formed Sulfmoc-Gly-Val-OH is released by eluting with 1M NaClsolution.

Example 18: Synthesis of Ile-Pro-Phe-OH

Smoc-Ile-OH is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Ile-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Ile iswashed twice with water. After the washing steps, 1.5 equivalents L-Prosolved in water are added to the activated Smoc-Ile and reacted at 25°C. for 12 minutes at pH 8.5 under agitation. Excess of L-Pro andreleased NHS are removed by washing with water. Smoc-Ile-Pro-OH isactivated by 2.5 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 1.2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume). Thereafter, the ion exchanger loaded with theactivated Smoc-Ile-Pro is washed twice with water. After the washingsteps, 1.5 equivalents L-Phe solved in water are added to the activatedSmoc-Ile-Pro and reacted at 25° C. for 12 minutes at pH 8.6 underagitation. Excess of L-Phe and released NHS are removed by washing withwater. The formed Ile-Pro-Phe-OH is released by deprotection using 1MNaOH (pH 9.5, 12 minutes) or Na₂CO₃ (pH 10, 25 minutes).

Example 19: Synthesis of Ile-Val-Phe-OH

Smoc-Ile-OH is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Ile-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Ile iswashed twice with water. After the washing steps, 1.5 equivalents L-Valsolved in water are added to the activated Smoc-Ile and reacted at 25°C. for 12 minutes at pH 8.5 under agitation. Excess of L-Val andreleased NHS are removed by washing with water. Smoc-Ile-Val-OH isactivated by 2.5 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 1.2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume). Thereafter, the ion exchanger loaded with theactivated Smoc-Ile-Val is washed twice with water. After the washingsteps, 1.5 equivalents L-Phe solved in water are added to the activatedSmoc-Ile-Val and reacted at 25° C. for 12 minutes at pH 8.6 underagitation. Excess of L-Phe and released NHS are removed by washing withwater. The formed Ile-Val-Phe-OH is released by deprotection using 1MNaOH (pH 9.5, 12 minutes) or Na₂CO₃ (pH 10, 25 minutes).

Example 20: Synthesis of Ile-His-Ile-OH

Smoc-Ile-OH is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Ile-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Ile iswashed twice with water. After the washing steps, 1.5 equivalents L-Hissolved in water are added to the activated Smoc-Ile and reacted at 25°C. for 10 minutes at pH 8.2 under agitation. Excess of L-His andreleased NHS are removed by washing with water. Smoc-Ile-His-OH isactivated by 2.5 equivalents2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline in mixture of water andacetonitrile (MeCN) 3:1 ratio (volume). Thereafter, the ion exchangerloaded with the activated Smoc-Ile-His is washed twice with water. Afterthe washing steps, 1.5 equivalents L-Ile solved in water are added tothe activated Smoc-Ile-His and reacted at 25° C. for 10 minutes at pH8.5 under agitation. Excess of L-Ile and released 1,2-dihydroquinolineand ethanol are removed by washing with water. The formed Ile-His-Ile-OHis released by deprotection using 1M NaOH (pH 9.5, 12 minutes) or Na₂CO₃(pH 10, 25 minutes).

Example 21: Synthesis of Leu-Ile-His-OH

Smoc-Ile-OH is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Ile-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume).

The formed Ile-His-OH is released by deprotection using organic baselike ethanolamine, N,N-diisopropylethylamine, triethylamine or 5%N-methyl-morpholine_((aq)). Ile-His is precipitated by adding aproticsolvents and washed till base residues are removed.

Thereafter, the ion exchanger is regenerated with 1M NaCl solution andwashed twice with water. Smoc-Ile-OH is added to the ion exchange resinat a maximum load and is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and acetonitrile (MeCN)3:1 ratio (volume). Thereafter, the ion exchanger loaded with theactivated Smoc-Leu is washed twice with water. After the washing steps,1 equivalents L-Ile-His solved in water are added to the activatedSmoc-Leu and reacted at 25° C. for 10 minutes at pH 8.5 under agitation.Released NHS is removed by washing with water. The formed Leu-Ile-His-OHis released by deprotection using 1M NaOH (pH 9.5, 12 minutes) or Na₂CO₃(pH 10, 25 minutes).

Example 22: Synthesis of LAGV-OH

Smoc-Ile-OH is added to an ion exchange resin (DEAE Sephadex A-25; GEHealthcare) at a maximum load. Smoc-Leu-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and isopropanole 2:1ratio (volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Ile iswashed with water. After the washing step, 1.5 equivalents L-Ala solvedin water are added to the activated Smoc-Leu and reacted at 25° C. for10 minutes at pH 7 under agitation. Excess of L-Ala and released NHS areremoved by washing with water. Smoc-Leu-Ala-OH is activated by 2.5equivalents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid and 2equivalents N-hydroxysuccinimide in mixture of water and isopropanole2:1 ratio (volume). Thereafter, the ion exchanger loaded with theactivated Smoc-Leu-Ala is washed twice with water. After the washingsteps, 1.5 equivalents Gly solved in water are added to the activatedSmoc-Leu-Ala and reacted at 25° C. for 10 minutes at pH 7 underagitation. Excess of Gly and released NHS are removed by washing withwater. Smoc-LAG-OH is activated by 2.5 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid and 2 equivalentsN-hydroxysuccinimide in mixture of water and isopropanole 2:1 ratio(volume). Thereafter, the ion exchanger loaded with the activatedSmoc-LAG is washed twice with water. After the washing steps, 1.5equivalents Val solved in water are added to the activated Smoc-LAG andreacted at 25° C. for 15 minutes at pH 7 under agitation. Excess of Valand released NHS are removed by washing with water.

The formed LAGV-OH is released by deprotection using NaOH (pH 10, 20minutes) or Na₂CO₃ (pH 10, 25 minutes). Yield: 20%.

Example 23: Synthesis of Leu-Enkephalin (YGGFL)

Smoc-Tyr-OH is obtained in a similar manner as disclosed above withregard to Smoc-ß-alanine (see Example 3). Smoc-Tyr-OH is added to an ionexchange resin (DEAE Sephadex A-25; GE Healthcare) at a maximum load.Smoc-Tyr-OH is activated by 3 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid and 2 equivalentsN-hydroxysuccinimide (NHS) in mixture of water and MeCN 2:1 ratio(volume).

Thereafter, the ion exchanger loaded with the activated Smoc-Tyr iswashed with water. After the washing step, 1.5 equivalents Gly solved inwater are added to the activated Smoc-Tyr and reacted at 25° C. for 10minutes at pH 7 under agitation. Excess of Gly and released NHS areremoved by washing with water. Smoc-YG-OH is activated by 2.5equivalents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid and 2equivalents N-hydroxysuccinimide in mixture of water and MeCN 2:1 ratio(volume). Thereafter, the ion exchanger loaded with the activatedSmoc-YG is washed with water. After the washing steps, 1.5 equivalentsGly solved in water are added to the activated Smoc-YG and reacted at25° C. for 10 minutes at pH 7 under agitation. Excess of Gly andreleased NHS are removed by washing with water. This Procedure isrepeated for each step until the complete sequence of Smoc-YGGFL-OH issynthesized.

The formed YGGFL-OH is released by deprotection using NaOH (pH 10, 20minutes) or Na₂CO₃ (pH 10, 25 minutes). Yield: 19%.

Example 24: Synthesis of Leu-Enkephalin (YGGFL) on SPPS

Tyr(tBu)-OH is added to an solid phase peptide resin (TentaGel® S TRT CIResin; 0.2-0.3 mmol/g) at a maximum load via the amine function.SolidS-Tyr(tBu)-OH is activated by 4 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid hydrochloride and 3equivalents Ethyl cyano(hydroxyimino)acetat (Oxyma) in mixture of waterand MeCN 2:1 ratio (volume).

Thereafter, the solid support loaded with the activated Tyr(tBu) iswashed with water. After the washing step, 3 equivalents Gly solved inwater are added to the activated SolidS-Tyr(tBu) and reacted at 25° C.for 30 minutes at pH 8 under agitation. Excess of Gly and released Oxymaare removed by washing with water. SolidS-YG-OH is activated by 4equivalents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimid hydrochlorideand 3 equivalents Ethyl cyano(hydroxyimino)acetat (Oxyma) in mixture ofwater and MeCN 2:1 ratio (volume). Thereafter, solid support loaded withthe activated Smoc-YG is washed with water. After the washing steps, 3equivalents Gly solved in water are added to the activated SolidS-YG andreacted at 25° C. for 30 minutes at pH 8 under agitation. Excess of Glyand released Oxyma are removed by washing with water. This procedure isrepeated for each step until the complete sequence of SolidS-YGGFL-OH issynthesized.

The formed YGGFL-OH is cleaved from solid support by using 95%trifluoroacetic acid (TFA) for 1.5 h. Yield: 30.5%.

Example 25: Synthesis of acetyl hexapeptide-3 (EEMQRR-OH) on SPPS

Glu(OtBu)-OH is added to an solid phase peptide resin (TentaGel® S TRTCI Resin; 0.2-0.3 mmol/g) at a maximum load via the amine function.SolidS-Glu(OtBu)-OH is activated by 4 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid hydrochloride and 3equivalents Ethyl cyano(hydroxyimino)acetat (Oxyma) in mixture of waterand MeCN 2:1 ratio (volume).

Thereafter, the solid support loaded with the activated Glu(OtBu) iswashed with water. After the washing step, 3 equivalents Glu(OtBu)solved in water are added to the activated SolidS-Glu(OtBu) and reactedat 25° C. for 30 minutes at pH 8 under agitation. Excess of Glu(OtBu)and released Oxyma are removed by washing with water. SolidS-EE-OH isactivated by 4 equivalents 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidhydrochloride and 3 equivalents Ethyl cyano(hydroxyimino)acetat (Oxyma)in mixture of water and MeCN 2:1 ratio (volume). Thereafter, solidsupport loaded with the activated SolidS-EE is washed with water. Afterthe washing steps, 3 equivalents Met solved in water are added to theactivated SolidS-EE and reacted at 25° C. for 30 minutes at pH 8 underagitation. Excess of Met and released Oxyma are removed by washing withwater. This procedure is repeated for each step until the completesequence of SolidS-EEMQRR-OH is synthesized. Arginine was used withoutside chain protecting groups.

The formed EEMQRR-OH is cleaved from solid support and side chaindeprotected by using 95% Trifluoroacetic acid (TFA) for 1.5 h. Yield:32%.

Example 26: Synthesis of deca-Ala on SPPS

Ala-OH is added to an solid phase peptide resin (TentaGel® S TRT CIResin; 0.2-0.3 mmol/g) at a maximum load via the amine function.SolidS-Ala-OH is activated by 4 equivalents1-ethyl-3-(3-dimethylaminopropyl)carbodiimid hydrochloride and 3equivalents Ethyl cyano(hydroxyimino)acetat (Oxyma) in mixture of waterand MeCN 2:1 ratio (volume).

Thereafter, the solid support loaded with the activated SolidS-Al iswashed with water two times. After the washing steps, 3 equivalents Alasolved in water are added to the activated SolidS-Ala-OH and reacted at25° C. for 30 minutes at pH 8 under agitation. Excess of Ala andreleased Oxyma are removed by washing with water. SolidS-AA-OH isactivated by 4 equivalents 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidhydrochloride and 3 equivalents Ethyl cyano(hydroxyimino)acetat (Oxyma)in mixture of water and MeCN 2:1 ratio (volume). Thereafter, solidsupport loaded with the activated SolidS-AA is washed with water. Afterthe washing steps, 3 equivalents Ala solved in water are added to theactivated SolidS-AA and reacted at 25° C. for 30 minutes at pH 8 underagitation. Excess of Ala and released Oxyma are removed by washing withwater. This procedure is repeated for each step until the completesequence of SolidS-decaAla-OH is synthesized.

The formed Deca-Ala-OH is cleaved from solid support by using 95%Trifluoroacetic acid (TFA) for 1.5 h. Yield: 17%.

Example 27: Synthesis of L-carnosine

4-(((L-histidyl)oxy)methyl)-N,N,N-trimethylbenzenaminium is added to anion exchange resin (SP Sephadex C-25; GE Healthcare) at a maximum load.Smoc-R-alanine is activated by 3 equivalent1-ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) and 2 equivalents Nhydroxysuccinimide (NHS) in mixture of water and isopropanol 4:1 ratio(volume). Thereafter the activated Smoc-ß-alanine-NHS ester is added toHistidine on the ion exchange column. The mixture is allowed to reactfor 15 min at pH 7.5 under agitation. Thereafter, the ion exchangerloaded with the Smoc-ß-Ala-His is washed two times with water to removethe excess of Smoc-ß-alanine and released NHS. Afterwards, theSmoc-protecting group is removed by 5% piperazine in water and washedtwo times with water. The formed L-carnosine is released by deprotectionusing 98% TFA

1. Method for preparing peptides, the method comprising a step offorming a peptide bond, wherein a carboxyl group of a first amino acidor first peptide is activated and an amino group of the first activatedamino acid or first peptide is protected and the activated carboxylgroup of the first amino acid or first peptide is reacted with an aminogroup of a second amino acid or second peptide wherein said carboxylgroup of the first amino acid or first peptide is activated in theabsence of the second amino acid or second peptide.
 2. Method accordingto claim 1, wherein said amino group of the first activated amino acidor first peptide is protected by a protecting group having awater-solubility enhancing group.
 3. Method according to claim 1,wherein said amino group of the first activated amino acid or firstpeptide is protected by a solid phase.
 4. Method according to claim 1,wherein a carboxyl group of the second amino acid or second peptidebeing reacted with said first activated amino acid or first peptide isnot protected.
 5. Method according to claim 1, wherein activation of thecarboxyl group of said first amino acid or first peptide and/or thereaction of the activated carboxyl group of the first amino acid orfirst peptide with the amino group of the second amino acid or secondpeptide is achieved using an environmentally friendly solvent.
 6. Methodaccording to claim 5, wherein said environmentally friendly solventcomprises a non-protic organic solvent and/or a secondary and/ortertiary alcohol.
 7. Method according to claim 1, wherein said formingof the peptide bond is achieved in solution having no strong basiccondition.
 8. Method according to claim 1, wherein said carboxyl groupof a first amino acid or first peptide is activated by a coupling agent.9. Method according to claim 1, wherein the first amino acid or firstpeptide is ionically contacted with an ion exchanger.
 10. Methodaccording to claim 1, wherein said forming of a peptide bond is achievedwhile an amino acid or a peptide is ionically bound to an ion exchangeror covalently bound to a solid phase.
 11. Method according to claim 1,wherein the carboxyl group of the first amino acid or first peptide isactivated by a coupling agent while the first amino acid or firstpeptide is ionically bound to an ion exchanger or covalently bound to asolid phase.
 12. Method according to claim 1, wherein the protection ofsaid amino acid or peptide is achieved by reacting an amino acid or apeptide with a protective agent comprising I. a backbone structure, II.at least one water-solubility enhancing group, and III. at least onereactive group, wherein the backbone structure comprises at least onemoiety selected from the group consisting of 9-methylfluorene, t-butaneand mono-, di- or triphenylmethane, wherein the water-solubilityenhancing group is selected from the group consisting of SO₃ ⁻, PO₃ ²⁻,N(CH₃)₂, N(CH₃)₃ ⁺, CN, OSO₃ ⁻ ester, OPO₃ ²⁻ ester, and combinationsthereof, and wherein the water-solubility enhancing group and thereactive group are attached to the backbone structure via at least onecovalent bond.
 13. Method according to claim 2, wherein said protectinggroup having a water-solubility enhancing group comprises at least twowater-solubility enhancing groups.
 14. Method according to claim 2,wherein said protecting group having a water-solubility enhancing groupcomprises exactly one water-solubility enhancing group.
 15. Methodaccording to claim 2, wherein the second amino acid or second peptidedoes not comprise any protecting groups with the exception of groupsprotecting a primary amine group.
 16. Peptide comprising a protectinggroup having a water-solubility enhancing group being bound to an aminogroup and an activated or free carboxyl group.
 17. Peptide according toclaim 16 being ionically bound to an ion exchanger.
 18. Method accordingto claim 6, wherein said environmentally friendly solvent comprises anon-protic organic solvent.
 19. Method according to claim 7, whereinsaid forming of the peptide bond is achieved in solution at a pH below10 or 12, measured by adding water to the solution at 25° C.
 20. Methodaccording to claim 7, wherein said forming of the peptide bond isachieved in solution at a pH value in the range of 4 to 12, or in a pHrange of 6 to 10, or in a pH range of 7 to 9.5, or in a pH range of 7 to9.0, or in a pH range of 7 to 8.5.
 21. Method according to claim 13,wherein said protecting group having a water-solubility enhancing groupcomprises at least two ionic groups.
 22. Peptide according to claim 16,comprising a protecting group having a water-solubility enhancing groupbeing bound to an amino group and an activated carboxyl group.